Cancer Cell International This Provisional PDF corresponds to the article as it appeared upon acceptance Fully formatted PDF and full text (HTML) versions will be made available soon In vitro activities of novel 4-HPR derivatives on a panel of rhabdoid and other tumor cell lines Cancer Cell International 2011, 11:34 doi:10.1186/1475-2867-11-34 Melissa E Smith (melissa.smith@phd.einstein.yu.edu) Bhaskar C Das (bhaskar.das@einstein.yu.edu) Ganjam V Kalpana (ganjam.kalpana@einstein.yu.edu) ISSN Article type 1475-2867 Primary research Submission date 22 April 2011 Acceptance date 27 September 2011 Publication date 27 September 2011 Article URL http://www.cancerci.com/content/11/1/34 This peer-reviewed article was published immediately upon acceptance It can be downloaded, printed and distributed freely for any purposes (see copyright notice below) Articles in Cancer Cell International are listed in PubMed and archived at PubMed Central For information about publishing your research in Cancer Cell International or any BioMed Central journal, go to http://www.cancerci.com/authors/instructions/ For information about other BioMed Central publications go to http://www.biomedcentral.com/ © 2011 Smith et al ; licensee BioMed Central Ltd This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited In vitro activities of novel 4-HPR derivatives on a panel of rhabdoid and other tumor cell lines Melissa E Smith1, Bhaskar C Das 2,3,4, and Ganjam V Kalpana1,4,5 Department of Genetics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY, 10461, USA Department of Nuclear Medicine, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY, 10461, USA Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY, 10461, USA Albert Einstein Cancer Center, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY, 10461, USA Department of Microbiology and Immunology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY, 10461, USA Correspondence to: Professor Ganjam V Kalpana, Departments of Genetics and Microbiology & Immunology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY, 10461, USA Phone: 1-718-430-2354; Email: ganjam.kalpana@einstein.yu.edu Email addresses: MES: melissa.smith@phd.einstein.yu.edu BCD: bhaskar.das@einstein.yu.edu GVK: ganjam.kalpana@einstein.yu.edu Abstract Background Rhabdoid tumors (RTs) are aggressive pediatric malignancies with poor prognosis N-(4-hydroxy phenyl) retinamide (4-HPR or fenretinide) is a potential chemotherapeutic for RTs with activity correlated to its ability to down-modulate Cyclin D1 Previously, we synthesized novel halogen-substituted and peptidomimeticderivatives of 4-HPR that retained activity in MON RT cells Here we analyzed the effect of 4-HPR in inhibiting the growth of several RT, glioma, and breast cancer cell lines and tested their effect on cell cycle, apoptosis and Cyclin D1 expression Methods Effect of compounds on RT cell cycle profiles, and cell death were assessed by MTS cell survival assays and FACS analysis The effects of treatment on Cyclin D1 expression were determined by immunoblotting The efficacy of these compounds on glioma and breast cancer cell lines was also determined using MTS assays Results Low micromolar concentrations of 4-HPR derivatives inhibited cell survival of all RT cells tested The 4-HPR derivatives altered RT cell cycle profiles and induced high levels of cell death that was correlated with their potency ATRA exhibited high IC50 values in all cell lines tested and did not cause cell death In MON RT cells, the iodosubstituted compounds were more active than 4-HPR in inducing cell cycle arrest and apoptosis Additionally, the activity of the compounds correlated with their ability to down-modulate Cyclin D1: while active compounds reduced Cyclin D1 levels, inactive ATRA did not In glioma and breast cancer cell lines, 4-HPR and 4-HPR derivatives showed variable efficacy Conclusions Here we demonstrate, for the first time, that the inhibitory activities of novel halogen-substituted and peptidomimetic derivatives of 4-HPR are correlated to their ability to induce cell death and down-modulate Cyclin D1 These 4-HPR derivatives showed varied potencies in breast cancer and glioma cell lines These data indicate that further studies are warranted on these derivatives of 4-HPR due to their low IC50s in RT cells These derivatives are of general interest, as conjugation of halogen radioisotopes such as 18 F, 124 I, or 131 I to 4-HPR will allow us to combine chemotherapy and radiotherapy with a single drug, and to perform PET/SPECT imaging studies in the future Background Rhabdoid tumors (RTs) are aggressive pediatric malignancies that occur within the kidneys, soft tissues, and central nervous system [1-4] No standard or effective treatment strategies are currently available for these tumors and prognosis remains poor with a two-year survival rate of 10-25% [1, 3-6] Therefore, there is a need to develop novel therapeutic strategies for RTs INI1 (hSNF5, BAF47, SMARCB1) is a tumor suppressor biallelically deleted in RTs We have previously demonstrated that Cyclin D1 is a key target repressed by INI1 and is required for genesis and survival of RTs in vitro and in vivo Furthermore, we have found that 4-HPR effectively inhibits RT growth and that the ability of 4-HPR to inhibit RTs is correlated to down-modulation of Cyclin D1 [7, 8] 4-HPR is a synthetic retinoid that is well tolerated by humans and inhibits the growth of various cancer cells by inducing apoptosis and, in some cases, cell cycle arrest at various stages [9-13] Inhibition of tumor cell growth by 4-HPR in vitro occurs at clinically achievable concentrations (IC50s ranging from µM to 15 µM) [14] 4-HPR is largely studied as a chemo-preventive agent in animal models of carcinogen-induced epithelial tumors and in patients at risk for breast cancer [10, 11, 15, 16] Additionally, treatment of pediatric neuroblastoma patients with 4-HPR has resulted in prolonged stabilization of disease in pilot clinical studies [17-20] 4-HPR induces apoptosis in tumor cells in vitro by various mechanisms including activating retinoic acid receptors (RAR) β and γ, inducing ceramide-dependent cytotoxicity, generating free radical oxygen species, inducing nitric oxide synthase expression resulting in nitric oxide-dependent cytotoxicity, and increasing the mitochondrial permeability transition [11, 15, 17, 18, 20] 4-HPR also induces cell cycle arrest likely correlated with its ability to down-modulate proliferation-related targets such as c-Myc, telomerase, p34/cdc2, and Cyclin D1 [21, 22] Interestingly, over expression of Cyclin D1 sensitizes breast cancer cells to 4-HPR [23] Based on this information, and the fact that RTs are dependent on Cyclin D1, we previously tested 4-HPR and found that it inhibits the growth of RTs in vitro and in vivo in a xenograft mouse model with efficacy correlated with down-modulation of Cyclin D1 [7] Several reports indicate that synthetic analogs of 4-HPR are more active antitumor agents or have potentially reduced toxicities compared to 4-HPR For example, N-benzyl hydroxyl retinamide, a non-hydrolysable carbon-linked analog does not suppress plasma vitamin A levels as 4-HPR does, and is therefore less toxic [24] Additionally, conjugations of 4-HPR have exhibited increase anti-tumor activity [25, 26] Because derivatives of 4-HPR may demonstrate more potent biological activity, improved bioavailability, and/or reduced toxicities compared to 4-HPR itself, we previously synthesized several novel 4-HPR derivatives which are conjugated to halogens and other moieties, and peptidomimetic derivatives that replace the alkene backbone with a rigid ring structure to help increase compound stability and bioavailability in vivo [27, 28] In particular, the peptidomimetic derivatives are more lipophilic, which increases bioavailability and possibly facilitates crossing the bloodbrain-barrier [29] Additionally, the derivatives that have been conjugated to halogens may allow us to create radio-conjugates (i.e 4-HPR conjugated to 18 F, 124 I, or 131 I) that facilitate combining radiotherapy and chemotherapy in a single agent In this study we examined the activity of 4-HPR derivatives that we synthesized in the laboratory and tested their activity on RT cell lines and investigated their abilities to induce apoptosis, cell cycle arrest, and down-modulate Cyclin D1 Additionally, we defined the activity profiles of these compounds in several glioma and breast cancer cell lines, for which 4-HPR has also been suggested as a potential chemotherapeutic agent We found that the 4-HPR derivatives tested potently inhibited RT cell survival with efficacy correlated with down-modulation of Cyclin D1 The derivatives tested were also active in inhibiting the growth of several glioma and breast cancer cell lines Our results indicate that because of their low IC50s and their abilities to significantly down-modulate Cyclin D1 and induce apoptosis, halogen-conjugated and peptidomimetic derivatives of 4-HPR are likely to be excellent diagnostic and therapeutic agents for RTs Additionally, the ability of the derivatives to significantly decrease the survival of glioma and breast cancer cell lines indicates that they may be of general interest for treating cancers that respond to 4-HPR or depend on Cyclin D1 for growth Results and Discussion Low micromolar concentrations of 4-HPR and its derivatives effectively inhibit rhabdoid tumor cell growth Our previous studies indicated that halogen-substituted and peptidomimetic derivatives of 4-HPR have activity against one RT cell line, namely, MON [27, 28] These studies neither elucidated the mechanism of action of these new compounds nor tested activities on a wide range of RT or non-RT cell lines Here we tested the efficacy of 4-HPR and highly active halogen-substituted (5h and 5j) and peptidomimetic derivatives (11a, 11c, and 11d) on three RT cell lines, MON, G401, and A204 (Figure 1) Since 4-HPR is derived from all trans retinoic acid (ATRA), which is used clinically as a differentiating and chemotherapeutic agent [30, 31], we used ATRA as a control The three cell lines cells were treated with increasing concentrations of indicated drugs and cell survival was determined We found that ATRA did not show any activity in reducing the survival of RT cells, indicating that this compound is not a useful candidate for RT therapy (IC50s>50 µM, Figure 2) 4-HPR potently inhibited the survival of RT cells with IC50s of 0.1 µM, 2.0 µM, and 2.2 µM in G401, A204, and MON cells respectively (Figure 2) Interestingly, iodo-derivatives of 4-HPR and the peptidomimetic compound tested, more potently decreased cell survival in MON cells with IC50s of 1.2 µM (5j) and 1.3 µM (11d) (Figure 2A) However, the derivatives and peptidomimetic compounds exhibited similar or less potent activities when compared to 4-HPR in G401 and A204 cells A summary of IC50s of all the compounds in these three different RT cell lines is listed in Table Our analysis indicated that the iodo-conjugated derivatives display significant potency in all RT cell lines tested Since iodo conjugation can potentially be used to deliver radiotherapy and taken advantage of for radiation-based imaging (by substituting with 124 I or 131I isotopes), these compounds are of great interest for further study Hence we tested the ability of these compounds to induce cell cycle arrest and apoptosis, and to down-modulate Cyclin D1 in RT cells Induction of cell cycle arrest in rhabdoid tumor cells lines treated with 4-HPR and its derivatives To determine the effect of 4-HPR derivatives on the cell cycle profiles of RT cells we carried out FACS analysis of cells treated with or 10 µM concentrations each of ATRA (as a control), 4-HPR, and its derivatives ATRA did not induce cell cycle arrest in MON, G401, or A204 cells, consistent with the observation that this drug is not active against RT cells (Figure 3) We found that treatment with 4-HPR and its derivatives, however, induced complex changes in the cell cycle profiles of these cells In MON cells, 4-HPR slightly increased the percentage of cells in S-phase, while both iododerivatives induced significant S-phase cell cycle arrest (P =0.0057 for µM 5j, P 50 6.9 18.9 12.0 10.4 15.7 3.8 >50 5.0 16.3 13.7 5.0 9.3 9.4 >50 1.4 5.8 9.1 2.4 5.9 7.8 Breast MCF7 MDAMB231 >50 2.2 6.3 11.8 8.3 12.1 8.2 *The IC50 could not be determined because the survival curve was not sigmoidal 24 >50 0.4 N/A* 23 2.3 5.3 12.0 Figure Figure Figure Figure Figure Figure Figure .. .In vitro activities of novel 4-HPR derivatives on a panel of rhabdoid and other tumor cell lines Melissa E Smith1, Bhaskar C Das 2,3,4, and Ganjam V Kalpana1,4,5 Department of Genetics, Albert... combined chemotherapy and radiotherapy Radioactive derivatives can also be used to evaluate bioavailability of 4-HPR in vivo, using non-invasive imaging In addition, the peptidomimetic derivatives. .. cancer cells by inducing apoptosis and, in some cases, cell cycle arrest at various stages [9-13] Inhibition of tumor cell growth by 4-HPR in vitro occurs at clinically achievable concentrations